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NDT Advance Access originally published online on January 31, 2006
Nephrology Dialysis Transplantation 2006 21(5):1289-1292; doi:10.1093/ndt/gfk072
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© The Author [2006]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Original Articles: Clinical Nephrology

Novel compound heterozygote mutations (H234Q/R1206X) of the ADAMTS13 gene in an adult patient with Upshaw–Schulman syndrome showing predominant episodes of repeated acute renal failure

Yugo Shibagaki1, Masanori Matsumoto2, Koichi Kokame3, Shigeyoshi Ohba1, Toshiyuki Miyata3, Yoshihiro Fujimura2 and Toshiro Fujita1

1 Department of Nephrology and Endocrinology, Graduate School of Medicine, University of Tokyo, Tokyo, 2 Department of Blood Transfusion Medicine, Nara Medical University, Nara and 3 National Cardiovascular Center Research Institute, Osaka, Japan

Correspondence and offprint requests to: Yugo Shibagaki, 7–3–1 Hongo Bunkyo-ku, Tokyo 113-8655, Japan. Email: eugo{at}wc4.so-net.ne.jp



   Abstract
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 Abstract
Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Unlike acquired thrombotic thrombocytopenic purpura or haemolytic uraemic syndrome, which are often intractable, thrombotic microangiopathy in patients with Upshaw–Schulman syndrome (USS) – a congenital deficiency of von Willebrand factor-cleaving protease (ADAMTS13) activity – responds very well to plasma infusion and does not even require plasma exchange. However, the symptoms significantly vary in each individual and thus clinicians often overlook this diagnosis.

Methods. A 31-year-old adult male patient with thrombotic microangiopathy, which was complicated with repeated episodes of acute renal failure, is reported. We suspected that the patient had USS and performed assays of ADAMT13 activity and its inhibitor, followed by ADAMTS13 gene analysis of the patient and his parents.

Results. The patient had extremely low ADAMTS13 activity and has no inhibitors of ADAMTS13. Through an ADAMTS13 gene analysis of this family, we found two novel mutations responsible for the disease: a missense mutation in exon 7 [702 C -> A (H234Q)] from the father and a nonsense mutation in exon 26 [3616 C -> T (R1206X)] from the mother.

Conclusions. Our experience appears to indicate the importance of assays of ADAMTS13 activity and its inhibitor in patients who have episodes of renal insufficiency in association with thrombotic microangiopathy, for diagnosis and choice of treatment.

Keywords: acute renal failure; genetic disorder; haemolytic uraemic syndrome; thrombotic microangiopathy; von Willebrand factor



   Introduction
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 Abstract
 Introduction
Subjects and methods
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Thrombotic thrombocytopenic purpura (TTP) and haemolytic uraemic syndrome (HUS) are both categorized within thrombotic microangiopathy (TMA), featured by microangiopathic haemolytic anaemia with thrombocytopenia [1]. The term TTP typically refers to a form of TMA that affects adolescents and adults and predominantly causes central nervous system disorders, whereas HUS refers to TMA, which mainly involves kidney and typically affects young children with diarrhoea caused by Escherichia coli O157:H7 infection. TTP and HUS are mostly indistinguishable by laboratory findings and pathology and thus in clinical practice are often referred to as HUS/TTP [2]. It has, however, been recognized that some forms of TTP respond well to plasma infusion (PI) [3] or plasma exchange (PE), whereas typical diarrhoea-associated HUS does not [4]. Recently, the plasma activity of von Willebrand factor (VWF)-cleaving protease (ADAMTS13, a disintegrin and metalloprotease domain, with thrombospondin type 1 motif 13) was found to be deficient in an inherited form of TTP, which differs from acquired idiopathic TTP characterized by its neutralizing or non-neutralizing inhibitor and from acquired HUS by subnormal enzyme activity [4–6]. If the onset of this inherited form of TTP is in the neonatal period, it is alternatively called Upshaw–Schulman syndrome (USS), although there is also an adult onset form of the disease [7].



   Subjects and methods
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 Subjects and methods
Results
 Discussion
 References
 
Patient
A 31-year-old male was admitted to the nephrology service for acute renal failure, microangiopathic haemolytic anaemia and thrombocytopenia.

He had a history of moderate jaundice as a newborn, which was treated with phototherapy. At 3 months of age, he had episodes of purpura and thrombocytopenia after diphtheria/pertussis/tetanus immunization, for which he was diagnosed with idiopathic thrombocytopenic purpura. Since the age of 2 years, he had an episode of intracranial bleeding after minor head injury and several episodes of high-renin hypertension and acute renal failure complicated with TMA, all of which were successfully treated with PI. Since then he has been treated with PI every other week to maintain blood platelet count >2 x 109/l, by which he was diagnosed by a previous paediatrician with recurrent HUS/TTP of unknown aetiology.

On admission, he showed no neurological deficits or any other specific symptoms, but subsequently his serum creatinine progressively rose from 0.87 to 5.78 mg/dl with increased LDH (1968 IU/l), low platelet count (3.5 x 109/l) and the presence of schistocytes. However, with PI, LDH rapidly fell back to normal level and his renal dysfunction and thrombocytopenia gradually resolved. He had no apparent family history of congenital coagulopathy, but his elder brother, who died at the age of 2 years, had laboratory data compatible with TMA, which raised our suspicion of the presence of hereditary HUS/TTP, such as USS.

We subsequently performed assays of ADAMTS13 activity, followed by ADAMTS13 gene analysis of the patient and his parents.

Assay of ADAMTS13 activity and its inhibitor
Plasma ADAMTS13 activity was assayed by the method of Furlan et al. [5] based on VWF multimer analysis, with a slight modification as described before [8]. The ADAMTS13 activity of pooled normal plasma was defined as 100%. The normal range of ADAMTS13 activity was 102±23% (mean±SD) [9].

The inhibitor activity against ADAMTS13 was measured as described by Furlan et al. [10] based on the Bethesda method [3]. One unit of inhibitor was defined as the amount that reduced the ADAMTS13 activity to 50% of the control.

ADAMTS13 gene analysis
After obtaining approval from the ethics committees of both the sample-collecting hospital and gene analysis institute, and informed consent, ADAMTS13 genes of the patient and his parents were analysed by DNA sequencing. All 29 exons and exon–intron boundary sites of the ADAMTS13 gene were amplified and sequenced as recently described [11].



   Results
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 Abstract
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 Subjects and methods
 Results
Discussion
 References
 
ADAMTS13 activity of the patient's plasma, which was obtained on admission (and just before PI), was <3% of the pooled normal plasma (Figures 1 and 2) and its inhibitor was undetectable (<0.5 Bethesda U/ml). Three novel mutations were identified (Figure 1). The patient had compound heterozygous mutations comprised of a missense mutation in exon 7 [702 C -> A (H234Q)] inherited from his father and a nonsense mutation in exon 26 [3616 C -> T (R1206X)] inherited from his mother. His father had an additional missense mutation at exon 21 [2708 C -> T (S903L)], but its effect on ADAMTS13 activity is presently unknown [11,12]. Thus, on this occasion, a solid diagnosis of USS was made. Although the patient's parents had heterozygous mutations and relatively low ADAMTS13 activity (father: 24%, mother: 21%; Figure 2), they had no episode of haematological problems and, to date, have been in good health. To investigate the frequencies of these mutations, we sequenced the relevant exons of ADAMTS13 in the general Japanese population. Seven heterozygotes carrying S903L were found in 64 subjects, suggesting that the mutation is a common polymorphism with an allele frequency of 5.5%. On the other hand, both H234Q and R1206X mutations were excluded as common polymorphisms by the screening of 64 subjects.


Figure 1
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  		Fig. 1. Pedigree of the index patient with gene haplotypes and plasma activity of ADAMTS13. The circle represents a female and squares represent males. Plasma ADAMTS13 activity (%) is shown under the circle and the squares. Mutations found in the ADAMTS13 gene are shown as one-letter amino acid abbreviations numbered from the initial Met codon. The arrow indicates the index patient. Both the mother and the father of the index patient are asymptomatic carriers. {dagger}A deceased individual. P, index patient; ND; value not determined.

 

Figure 2
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  		Fig. 2. Cleavage of VWF multimer by ADAMTS13. Quantitative assay of plasma ADAMTS13 activity from a control, the patient and the parents. Following incubation of VWF, plasma from six dilution series of a normal control and plasma from the patient and his parents, sodium dodecyl sulphate–agarose gel electrophoresis and western blotting were performed as shown in a previous report [8]. The plasma ADAMTS13 activity was shown as a percentage of the normal control.

 


   Discussion
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
References
 
USS, which may be alternatively called congenital chronic relapsing TTP or familial HUS/TTP, is characterized by repeated episodes of thrombocytopenia and haemolytic anaemia which ameliorate quickly by PI [13]. Typically, patients have episodes of hyperbilirubinaemia with negative Coombs test during infancy followed by repeated episodes of TTP [14]. In 1997, Furlan et al. [10] reported four cases of chronic relapsing TTP lacking ADAMTS13 activity. Since then, ADAMTS13 has been known to have a central pathogenic role in TTP and a defect in its gene was reported to cause USS, which is a form of TTP with a neonatal onset, although there is also an adult onset disease caused by ADAMTS13 gene defect [15,7]. Patients with USS either have homozygous or compound heterozygous gene mutations [11,12,15]. Another familial or inherited form of HUS/TTP is associated with factor H deficiency. Patients with factor H deficiency have, predominantly, renal impairment and low level of serum C3. Factor H deficiency seems to aggravate C3 activation, which potentiates autoantibody-mediated or immune complex-mediated glomerular injury. While some patients with factor H deficiency were reported to accompany low ADAMTS13 level and renal involvement [16], our case showed normal factor H level (data not shown).

In the present study, we identified two novel mutations, H234Q in exon 7 and R1206X in exon 26, in the ADAMTS13 gene. The missense mutation, H234Q, resides in the metalloprotease domain. The conserved active site sequence HEXXHXXGXXHD (amino acids 224–235) in this domain contains 3 His residue that coordinate the catalytic Zn2+ ion [17]. It is a reasonable assumption that if one His residue is replaced with Gln, the mutant lacks its protease activity. The nonsense mutation, R1206X, resides in the CUB1 domain of the ADAMTS13 gene. This mutant lacks 222 amino acids in the C-terminal that include a part of the CUB1 domain and the entire CUB2 domain. The deletion of these regions may cause secretion defects during synthesis. It has been suggested that the CUB domains are important for interactions between ADAMTS13 and VWF [18]. Therefore, deletion of the C-terminal region may affect substrate recognition. In the previous study, we sequenced exon 7 in 364 Japanese individuals and found no H234Q mutation [9]. The R1206X mutation was not observed in the 12 Japanese families with USS so far determined in our laboratory [11,12]. However, recombinant ADAMTS13 reproducing the patient's mutations has not been performed; the above-mentioned mechanisms of inducing ADAMTS13 deficiency are only speculative at this point.

Unusually large VWF multimers (UL-VWFMs) are secreted from vascular endothelial cells. UL-VWFMs tend to aggregate platelets to form UL-VWFM–platelet complexes, which may embolize microvessels and cause organ ischaemia. ADAMTS13 cleaves UL-VWFMs to a series of smaller multimers, which lack the tendency to aggregate platelets. Therefore, loss of ADAMTS13 activity caused either by genetic defects (USS) or by its inhibitors causes intravascular platelet aggregation, leading to TMA.

Thus, theoretically, early intervention with PE or PI should be effective in these disorders by supplementing ADAMTS13 with or without removal of its inhibitors, respectively. In fact, there has been evidence that extremely low levels of ADAMTS13 activity were associated with excellent response to PE and good prognosis [12,17], although there also are reports challenging this view [2]. Our experience of 20 patients with USS indicates that all are doing quite well with regular infusion of plasma, except one patient who died of TTP that became refractory to plasma after cholecystectomy.

Patients with TMA tend to be labelled as having TTP if clinical features are predominated by neurological dysfunction or HUS if renal impairment is the predominant feature. However, renal impairment accompanies as much as 44% of patients with TTP [19]. Even if the predominant feature of these patients with TMA is renal dysfunction, we need to consider diagnosis of TTP and check ADAMTS13 activity, especially if an inherited form of the disease is suspected. If ADAMTS13 activity is very low, PE in patients with inhibitors or PI in cases with USS would be justified and should be done as early as possible. Tsai has proposed that the definition of TTP should be a pro-thrombotic state in the microvasculature caused by severe ADAMTS13 deficiency, such that we can easily identify a group of patients who have a common pathogenetic mechanism for the future enzyme replacement or gene therapy [4]. Although some challenge this notion, it is still important to develop rapid and simple assay methods for ADAMTS13 activity so that the appropriate therapy can be given immediately.

Our experience appears to indicate that assays of ADAMTS13 activity and its inhibitor should be considered in patients who have repeated episodes of renal insufficiency as well as thrombocytopenia.



   Acknowledgments
 
The case of the patient was presented briefly in a review paper published in Hypertension Research journal [20].

Conflict of interest statement. None declared.



   References
Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 2. 8.05
Accepted in revised form: 22.12.05


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